The nature of human alkaline phosphatase polymorphism will be defined at the molecular level using a combination of techniques including monoclonal antibody binding, proteolytic digestion, and amino acid sequence determination of peptides differing among the placental alkaline phosphatase (PLAP) variants. In addition, the domain structure of alkaline phosphatase will be studied. The orientation of placental alkaline phosphatase with respect to the cell membrane has now been defined by proteolysis. It is known that bromelain releases PLAP from the cell surface by cleaving a 2,000 dalton membrane insertion piece containing around 20 amino acids. Under nondenaturing conditions, trypsin causes a decrease in molecular weight of each PLAP chain by 10,000. Autoradiographs of SDS gels in which tandem combinations of proteolytic digests of I-125 PLAP had been electrophoresed indicate that trypsin and bromelain cleave at opposite ends of the PLAP polypeptide chain. Two common phenotypes of PLAP with the same molecular weight and distinguished by electrophoresis are FF (Fast) and SS (Slow). Is the difference due to charged groups (terminal sialic acids), to amino acid composition and sequence or to conformation and shape? Removal of terminal sialic acid by neuraminidase does not change the relative migration of FF and SS. The pure FF and SS PLAP molecules were rotary shadowed on mica with platinum and were found to measure approximately 9 x 6 nm. Accordingly, the difference in electrophoretic migration is most likely due to amino acid composition and sequence. It is predicted that results such as these will contribute to filling the gap in information on the structure of placental alkaline phosphatase and its genetic and cancer-related variants.